Preparation of Magnetic Iron Oxide Nanoparticles (Mions) with Improved Saturation Magnetization Using Multifunctional Polymer Ligand

polymers

Article Preparation of Magnetic Iron Oxide Nanoparticles (MIONs) with Improved Saturation Magnetization Using Multifunctional Polymer Ligand

Muhammad Irfan Majeed 1,†,‡, Jiaojiao Guo 1,2,†, Wei Yan 1,2,* and Bien Tan 1

1 School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China; [email protected] (M.I.M.); [email protected] (J.G.); [email protected] (B.T.) 2 Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry of Education, Key Laboratory of Green Preparation and Application for Functional Materials, Hubei Key Laboratory of Polymer Materials, School of Materials Science & Engineering, Hubei University, Wuhan 430062, China * Correspondence: [email protected]; Tel.: +86-27-8755-8172 † These authors contributed equally to this work. ‡ Current address: Department of Chemistry, University of Agriculture Faisalabad, Faisalabad 38040, Pakistan.

Academic Editors: Joannis K. Kallitsis, Georgios Bokias and Valadoula Deimede Received: 4 October 2016; Accepted: 31 October 2016; Published: 8 November 2016

Abstract: This paper describes the preparation of ultra-small magnetic iron oxide (Fe3O4) nanoparticles (MIONs) coated with water-soluble thioether end-functionalized polymer ligand pentaerythritol tetrakis 3-mercaptopropionate-polymethacrylic acid (PTMP-PMAA). The MIONs were prepared by co-precipitation of aqueous iron precursor solution at a high temperature. The polymer modified MIONs were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), thermogravimetric analysis (TGA), and vibrating sample magnetometery (VSM). It was found that these MIONs were successfully modified by this water-soluble polymer ligand with a fairly uniform size and narrow size distribution. The dried powder of MIONs could be stored for a long time and re-dispersed well in water without any significant change. Additionally, the polymer concentration showed a significant effect on size and magnetic properties of the MIONs. The saturation magnetization was increased by optimizing the polymer concentration. Furthermore, the 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT)-assay demonstrated that these MIONs were highly biocompatible and they could be successfully coupled with fluorescent dye Rhodamine due to the formation of amide bond between carboxylic acid groups of MIONs and amine groups of dye. The obtained results indicated that these multifunctional MIONs with rich surface chemistry exhibit admirable potential in biomedical applications.

Keywords: magnetic iron oxide nanoparticles; polymer ligand; biocompatible; multifunctional; saturation magnetization

1. Introduction Magnetic nanoparticles (NPs) have gained much scientiﬁc interest for their unique magnetic properties such as superparamagnetism, high coercivity, low Curie temperature, and high magnetic susceptibility [1,2]. Magnetic iron oxide nanoparticles (MIONs), owing to their advantages such as low toxicity and biocompatibility, are considered to be the most favorable candidates for bio-applications [3] including magnetic resonance imaging (MRI) [4,5], magnetic ﬂuid hyperthermia (MFH) [6,7], magnetic separation and immobilization of biomolecules such as nucleic acids and proteins [8,9],

Polymers 2016, 8, 392; doi:10.3390/polym8110392 www.mdpi.com/journal/polymers Polymers 2016, 8, 392 2 of 16 the development of drug delivery systems for controlled release of drugs [10,11], biolabeling and magnetic sensors [12]. Thermal decomposition and co-precipitation are among the most common techniques employed for MIONs preparation but both having advantages and limitations [13]. It is usually desired to develop a simple, one step, cost effective and environment friendly protocol for the preparation of water-soluble, uniform, multifunctional, superparamagnetic iron oxide NPs with a better control over their size, shape and magnetic properties. Furthermore, for bioapplications of MIONs, they should be highly dispersed in aqueous phase and biocompatible having least or no toxicity, which can be achieved through adopting co-precipitation method using multifunctional water-soluble polymers as capping ligands. Multifunctional water-soluble polymers as capping ligands can play an important role in the preparation of MIONs and other inorganic NPs [14–17]. In aqueous co-precipitation process, polymer ligands can efficiently control the size and shape of the NPs due to the presence of abundant functional groups such as –COOH, –OH, –NH2, etc. These multifunctional polymers render magnetic NPs more stable, water-soluble and uniform in addition to providing them rich surface chemistry which opens up ways for easier post-synthesis modification and functionalization for bio-applications [5,18–22]. Numerous studies have been reported for the synthesis of MIONs using polymer ligands in order to render them water-soluble and biocompatible. However, in most of the cases, the NPs are first prepared through thermal decomposition and then made water-soluble through post-synthesis modification processes such as ligand exchange using water-soluble polymer ligands [23,24]. In this study, we have described the synthesis of MIONs with a multifunctional water-soluble polymer ligand pentaerythritol tetrakis 3-mercaptopropionate-polymethacrylic acid (PTMP-PMAA), following our previous work [20]. Previously, it was demonstrated that PTMP-PMAA, having abundance of carboxylic acid groups, can be successfully used for the stabilization of the MIONs in the co-precipitation procedure. These carboxylic acid groups have ability to cap the growing MIONs in the reaction mixture through coordinating with iron oxide surface and thus stabilize them and control their size and size distribution depending upon the concentration of the polymer ligand. Therefore, the concentration of the polymer ligand or the molar ratio between carboxylic acid groups of the PTMP-PMAA and iron precursors play an important role in preparation of uniform MIONs through aqueous co-precipitation procedure. However, in that report, MIONs prepared with 0.768 mM concentration of PTMP-PMAA had very small size (4.5 ± 0.4 nm) and lower saturation magnetization (45 emu·g−1). Those MIONs, due to their high dispersibility and ultra-small size, were successfully used as dual MRI contrast agents [5]. However, due to their lower saturation magnetization, they could not be manipulated in dispersed state with the use of an external magnet, which limits their scope of applications. Therefore, herein, we report the preparation of MIONs using PTMP-PMAA with improved saturation magnetization through our original high temperature single step co-precipitation method [20] but with few modifications in order to improve the magnetic properties of MIONs, such as lower polymer concentrations as compared to previous report, iron precursors were dissolved in concentrated hydrochloric acid (HCl) instead of 1 M HCl solution in order to prevent their hydrolysis and condensation before the addition of precipitating agents and the inert conditions were maintained, by nitrogen gas bubbling throughout the course of reaction, which not only protects MIONs from critical oxidation but also keeps their size smaller [25]. MIONs prepared using PTMP-PMAA had several carboxylic acid (–COOH) functional groups which provide them excellent dispersibility and stability in aqueous solutions. These MIONs showed high resistance against aggregation in aqueous media over a wide range of pH and salt concentration due to excellent electrostatic and steric stabilization provided by the polymer ligand. These MIONs can be dried by evaporating solvent and stored as powder for several months without any undesired changes in their chemical and physical properties. The polymer ligand offers MIONs better chemical stability against oxidation which otherwise leads to a decrease in their magnetic properties. Furthermore, cytotoxicity analysis of the MIONs proved them to be biocompatible even at their high Polymers 2016, 8, 392 3 of 16 Polymers 2016, 8, 392 3 of 16 successfully conjugation of MIONs with a fluorescent dye Rhodamine (Rh 110). Such bimodal detection systems based on fluorescent molecules and magnetic NPs can facilitate the deep tissue concentration up to 500 µg·mL−1. Bio-applicability of these NPs was demonstrated by successfully imaging by combined optical and MRI techniques [26]. The fabrication of novel targeted conjugation of MIONs with a fluorescent dye Rhodamine (Rh 110). Such bimodal detection systems luminescent and magnetic NPs with multifunctional water-soluble polymers would play a vital role based on fluorescent molecules and magnetic NPs can facilitate the deep tissue imaging by combined in the development of contrast agents for diagnosis, imaging and therapeutic technologies of the optical and MRI techniques [26]. The fabrication of novel targeted luminescent and magnetic NPs new era [26,27]. Finally, it was concluded that MIONs stabilized with PTMP-PMAA were extremely with multifunctional water-soluble polymers would play a vital role in the development of contrast biocompatible and can be used for several bio-applications due to their chemically rich surface agents for diagnosis, imaging and therapeutic technologies of the new era [26,27]. Finally, it was providing numerous opportunities for their conjugation with a variety of therapeutic, targeting, and concluded that MIONs stabilized with PTMP-PMAA were extremely biocompatible and can be used labeling agents. for several bio-applications due to their chemically rich surface providing numerous opportunities for 2.their Materials conjugation and Methods with a variety of therapeutic, targeting, and labeling agents. 2. Materials and Methods 2.1. Materials 2.1. MaterialsAll chemicals were of analytical grade and were used as received without any further purification,All chemicals unless were otherwise of analytical described. grade Methacrylic and were used acid as received(MAA, 99%), without 2,2 any′-azobisisobutyronitrile further purification, (AIBN,unless otherwise98%), anhydrous described. ethanol, Methacrylic anhydrous acid acetone (MAA, 99%),and anhydrous 2,20-azobisisobutyronitrile diethyl ether were (AIBN, purchased 98%), fromanhydrous National ethanol, Medicines anhydrous Corporation acetone and Ltd. anhydrous of China diethyl (Beijing, ether China). were purchasedPentaerythritol from Nationaltetrakis 3-mercaptopropionateMedicines Corporation (PTMP, Ltd. of China 99%) (Beijing,was obtained China). from Pentaerythritol Aldrich (St. tetrakis Louis, 3-mercaptopropionate MO, USA). Ferric 3 2 4 2 (PTMP,Chloride 99%) (FeCl was·6H obtainedO, 99%), ferrous from Aldrich sulfate (St.(FeSO Louis,·7H O, MO, 99%), USA). hydrochloric Ferric Chloride acid (HCl, (FeCl 38%)3·6H and2O, ammonium hydroxide (NH4OH, 28%) were obtained from Sinopharm Chemical Reagent Co. Ltd. 99%), ferrous sulfate (FeSO4·7H2O, 99%), hydrochloric acid (HCl, 38%) and ammonium hydroxide (Shanghai, China) N-(3-dimethylamino-propyl)-N′-ethylcarbodiimide hydrochloride (EDC) and (NH4OH, 28%) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China) N-hydroxysuccinimde-(3-dimethylamino-propyl)- (NHS)N were0-ethylcarbodiimide purchased from hydrochloride Aladdin Reagent (EDC) Co., and Ltd.N-hydroxysuccinimde (Shanghai, China). (NHS)Fetal bovine were purchasedserum (FBS) from and Aladdin Dulbecco’s Reagent modifi Co.,ed Ltd. eagle’s (Shanghai, medium China). (DMEM) Fetal were bovine obtained serum (FBS)from Gibcoand Dulbecco’s (Basel, Switzerland). modified eagle’s The 3-(4,5-Dime-ltetrazolium medium (DMEM) were bromide) obtained (MTT) from Gibco cell proliferation (Basel, Switzerland). assay kit Thewas 3-(4,5-Dime-ltetrazoliumreceived from Amresco bromide)(Solon, OH, (MTT) USA). cell proliferationMilli-Q water assay was kit used was in received the preparation from Amresco and (Solon,subsequent OH, application USA). Milli-Q experiments. water was used in the preparation and subsequent application experiments.

2.2. Synthesis and Characterizatio Characterizationn of Polymer Ligand PTMP-PMAA PTMP-PMAA was synthesized by free radical polymerization of monomermonomer methacrylic acid (MAA) using pentaerythritol tetrakis 3-mercaptopr 3-mercaptopropionateopionate (PTMP) as chain transfer agent as described in previous reports [[28–30].28–30]. The molar ratioratio of monomer to chain transfer agent helped to control the molecular weight of the polymer. SchemeScheme1 1 represents represents the the polymer polymer synthesis synthesis process. process.

Scheme 1. Synthesis of polymerpolymer ligandligand pentaerythritolpentaerythritol tetrakistetrakis 3-mercaptopropionate-polymethacrylic3-mercaptopropionate-polymethacrylic acid (PTMP-PMAA).

In a typical preparation for PTMP-PMAA, methacrylic acid (MAA, 5 g, 58 58 mmol), mmol), pentaerythritol tetrakis 3-mercaptopropionate (PTMP, (PTMP, 0.56 g, 1.16 mmol, 2% of monomer) and 2,2′0-azobisisobutyronitrile-azobisisobutyronitrile (AIBN, (AIBN, 0. 0.095095 g, 0.58 mmol, 1% 1% of of monomer) monomer) were were added added to to EtOH EtOH (25 (25 mL) mL) in a three-necked round-bottomed flask, flask, equipped equipped with with a a reflux reflux condenser condenser and and mechanical mechanical stirrer. stirrer. The temperaturetemperature of of the the reaction reaction mixture mixture was was maintained maintained at 75 at◦C 75 for °C 5 h for under 5 h Nitrogen under Nitrogen with vigorous with vigorousstirring. Atstirring. the end At of the this end period, of this the period, reaction the mixture reaction was mixture left to coolwas downleft to tocool room down temperature to room temperatureand then the and products then the were products isolated were by precipitation isolated by intoprecipitation cold diethyl into ether. cold diethyl The polymer ether. wasThe polymercollected was by filtration collected on by a filtration Buchner funnel,on a Buchner and the funnel, solvent and and the monomer solvent residuesand monomer were removedresidues

Polymers 2016, 8, 392 4 of 16 Polymers 2016, 8, 392 4 of 16 bywere evaporation removed toby constantevaporation mass to constant using a vacuummass using oven a vacuum set at 45 oven◦C. set A fractionat 45 °C. ofA fraction low molar of low mass polymer,molar mass un-reacted polymer, monomer un-reacted and somemonomer oligomers and remainingsome oligomers after reactionremaining are removedafter reaction during are the precipitationremoved during step. the The precipitation yield obtained step. was The 80% yield for obtained this reaction. was 80%1H-NMR for this spectra reaction. were 1H-NMR recorded spectra were recorded on a 400 MHz Bruker AV400 spectrometer (Billerica, MA, USA) using on a 400 MHz Bruker AV400 spectrometer (Billerica, MA, USA) using d6-DMSO as a solvent in ad 56 mm-DMSOquartz as a NMRsolvent tube in a at 5 roommm quartz temperature NMR tube using at theroomδ scaletemperature and were using consistent the δ scale to the and previous were reportsconsistent [28,29 to]. the The previous molecular reports weights [28,29]. of polymer The molecular were determined weights of bypolymer gel permeation were determined chromatography by gel (GPC)permeation on Agilent chromatography 1100 instrument (GPC) (Santaon Agilent Clara, 1100 CA, instrument USA) using (Santa THF Clara, as CA, mobile USA) phase using after THF its methylationas mobile phase with after TMS-diazomethane. its methylation with TMS-diazomethane.

2.3.2.3. Synthesis Synthesis of of MIONs MIONs MIONsMIONs synthesis synthesis processprocess waswas adopted from from our our pr previousevious work work with with few few modifications modiﬁcations [20,25]. [20,25 ]. Typical procedure involves the co-precipitation of aqueous iron precursor solution with ammonia in Typical procedure involves the co-precipitation of aqueous iron precursor solution with ammonia the presence of polymer ligand at high temperature (Scheme 2). Brief experimental details of the in the presence of polymer ligand at high temperature (Scheme2). Brief experimental details of the MIONs synthesis using polymer ligand PTMP-PMAA are provided in the following paragraph. MIONs synthesis using polymer ligand PTMP-PMAA are provided in the following paragraph.

SchemeScheme 2. 2.Graphical Graphical representation representation of of the the synthesis synthesi ofs PTMP-PMAAof PTMP-PMAA functionalized functionalized MIONs MIONs and and their subsequenttheir subsequent functionalization functionalization with Rhodaminewith Rhodamine 110. 110.

Briefly, in a 500 mL four-necked round-bottom flask equipped with reflux condenser, Briefly, in a 500 mL four-necked round-bottom flask equipped with reflux condenser, thermometer thermometer and nitrogen supply, 300 mL of Milli-Q water was added. The water was purged with andnitrogen nitrogen gas supply, to remove 300 mLoxygen of Milli-Q and was water heated was to added. reflux Thein oil water bath was with purged magnetic with stirring. nitrogen When gas to removetemperature oxygen reached and was 80 heated°C, the topolymer reflux inligand oil bath (PTMP-PMAA) with magnetic was stirring. introduced When to temperature flask to make reached the ◦ 80aqueousC, the solution polymer of ligand polymer (PTMP-PMAA) ligand (0.072 mM, was pH introduced = 4). When to the flask temperature to make the reached aqueous at 100 solution °C, the of ◦ polymeriron precursor ligand solution (0.072 mM, comprising pH = 4).of FeCl When3·6H the2O (3.24 temperature mmol) and reached FeSO4·6H at 1002O (1.62C, mmol) the iron in precursor6 mL of solutionconcentration comprising HCl was of FeCl added3·6H and2O (3.24the 90 mmol) mL of and concentration FeSO4·6H2 NHO (1.624OH mmol) was added in 6 mL within of concentration 5 s. Upon HCl was added and the 90 mL of concentration NH4OH was added within 5 s. Upon addition of iron

Polymers 2016, 8, 392 5 of 16 precursor solution, the color of the reaction mixture became yellow and upon addition of ammonia solution the color of the reaction mixture turned to dark black suddenly indicating the formation of iron oxide NPs. The temperature of the reaction mixture dropped to 85 ◦C upon the addition of iron precursor and ammonia addition and it took ~15 min to raise the temperature to 100 ◦C again and then the reaction was allowed to continue for 2 h at this temperature with vigorous magnetic stirring and constant nitrogen bubbling. After 2 h the heating was stopped and reaction mixture allowed cooling down to room temperature under nitrogen and then solvent was removed by rotary evaporator and solution concentrated to 60 mL and dialyzed against Milli-Q water for 72 h using dialysis membrane with molecular weight cut-off value 14,000 kDa. The dried NPs powder was obtained by evaporation of dialyzed NPs black suspension using rotary evaporator, washing with acetone and then drying in vacuum oven to a constant weight. The yield obtained was ~90%. Different samples of MIONs were prepared in the same way but with different concentrations of polymer ligand PTMP-PMAA, in order to obtain MIONs with optimum size and magnetic properties.

2.4. Characterization of MIONs The particle size of the MIONs was determined by a high performance particle sizer (Brookehaven Nano DLS 90Plus/BI-MAS, Holtsville, NY, USA) with multi angle particle sizing option (from Brookhaven Instruments Co., Holtsville, NY, USA) with an effective detection capability of 0.6 to 6000 nm. The zeta potential values were determined by a Brookhaven ZetaPlus (Brookhaven Instruments Co., Holtsville, NY, USA) at 25 ◦C using the folded capillary cells. Data were obtained using a monomodal acquisition and was fit according to the Smoluchowski theory. After filtration with aqueous membrane (Φ = 13 mm, 0.22 µm), the samples were analyzed using ultrapure water as solvent (pH = 7). These measurements were run at least three times with independent particle batches. Transmission electron microscopy images were recorded on a JEOL-2100 electron microscope (Akishima, Tokyo, Japan) operating at an acceleration voltage of 200 kV. TEM samples were prepared by the slow evaporation of a drop of dilute aqueous solution of MIONs onto carbon-coated copper grids (400 mesh). Images were recorded with a Gatan 794 CCD camera (Pleasanton, CA, USA). Size distribution graphs were prepared by analyzing about 200 individual NPs on each TEM image by ImageJ software. X-ray diffraction (XRD) was recorded on X’Pert PRO XRD spectrometer (PANalytical B.V., Kassel, Holand) using Cu Kα (λ = 1.54056 Å) radiation in the 2θ range of 10◦–80◦. The UV–vis spectra were recorded using Lambda 35 UV–vis spectrophotometer (Perkin-Elmer, Waltham, MA, USA). The infrared spectra were recorded by a Fourier transform infrared (FTIR) spectrometer (Bruker Vertex 70, Billerica, MA, USA) equipped with an attenuated total reflection (ATR) accessory. Raman spectra were measured on a HR JOBIN YVON spectrometer using a laser of 632 nm and a 25% filter. Thermogravimetric analysis (TGA) measurements were made using TGA Q500 (TA Instruments, New Castle, DE, USA) at a heating rate of 10 ◦C·min−1 from room temperature to 900 ◦C in oxygen free atmosphere. The saturation magnetizations (Ms) of the MIONs were measured at 26.8 ◦C on a Lakeshore 7400 Series vibrating sample magnetometer (Lake Shore Cryotonics, Westerville, OH, USA). All the magnetization data were normalized to the same weight.

2.5. Cytotoxicity Analysis of MIONs Toxicity analysis of the MIONs was carried out by MTT-assay using HepG2 cells. HepG2 cells were cultured in Dulbecco’s Modiﬁed Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 µg·mL−1 streptomycin and 100 U·mL−1 penicillin, in a humidiﬁed incubator at 37 ◦C with a 5% CO2 atmosphere. Cell viability was determined by MTT-assay. HepG2 cells were seeded into a 96-well plate with a cell density of 1 × 104 cells per well and suspended in DMEM supplemented ◦ with 10% FBS and incubated for 24 h at 37 C in a 5% CO2 atmosphere. After that, the cell culture medium was replaced with fresh medium containing different concentrations of (25, 50, 100, 200, 500 and 1,000 µg·mL−1) MIONs, PTMP-PMAA and MIONs@PTMP-PMAA in triplicate. A control experiment with only cell culture medium without any NPs or polymer was also carried out in each Polymers 2016, 8, 392 6 of 16

◦ case. Plates were placed at 37 C in a humidiﬁed 5% CO2 incubator and MTT-assay was performed after 24, 48 and 72 h. Polymers 2016, 8, 392 6 of 16 For MTT-assay, brieﬂy cell culture media were aspirated and 20 µL MTT (5 mg·mL−1)

[3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumin each case. Plates were placed at 37 °C in a humidified bromide] 5% inCO FBS2 incubator free DMEM and MTT-assay was added was to each ◦ well andperformed incubated after for24, another48 and 72 night h. at 37 C in a humidiﬁed 5% CO2 incubator. After incubation, MTT solution wasFor removedMTT-assay, and briefly 150 µ Lcell DMSO culture was media added were in eachaspirated vial to and dissolve 20 µL newly MTT formed(5 mg·mL formazan−1) crystals.[3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium The plates were placed on a swing bed for bromide] 10 min andin FBS then free absorbance DMEM was was added recorded to at 490 nmeach using well a and micro incubated plate reader for another (Thermo night Electron at 37 Corporation, °C in a humidified Waltham, 5% MA, CO2 USA). incubator. Theabsorbance After was recordedincubation, in MTT triplicate solution in eachwas removed case with and subtraction 150 µL DM forSO platewas added absorbance in each atvial 650 to nmdissolve and percentagenewly formed formazan crystals. The plates were placed on a swing bed for 10 min and then absorbance cell viability was calculated as the ratio of mean absorbance of triplicate readings with respect to mean was recorded at 490 nm using a micro plate reader (Thermo Electron Corporation, Waltham, MA, absorbance of control wells: USA). The absorbance was recorded in triplicate in each case with subtraction for plate absorbance at 650 nm and percentage cell viability was calculated as the ratio of mean absorbance of triplicate × readings with respect to meanCell absorbance viability of = control (Isample wells:/Icontrol ) 100

2.6. Conjugation of MIONs@PTMP-PMAACell viability with Rhodamine = (Isample/Icontrol 110) × 100

MIONs@PTMP-PMAA,2.6. Conjugation of MIONs@PTMP-PMAA EDC/NHS, and with Rh Rhodamine 110 were 110 separately dissolved in phosphate buffered · −1 saline (PBS)MIONs@PTMP-PMAA, solution (1 mL, pH EDC/NHS, = 6.8). The and concentration Rh 110 we ofre Rh separately 110 was 0.1dissolved and 1.0 in mg phosphatemL for the otherbuffered reagents. saline EDC (PBS) (1.45 solution mL) and (1 mL, NHS pH (400 = 6.8).µL) The were concentration sequentially of Rh added 110 was to Fe0.13 andO4 solution1.0 mg·mL (10−1 mL) and thenfor the Rh other 110 solutionreagents. (2.8 EDC mL) (1.45 was mL) added and NH andS (400 the mixtureµL) were was sequentially stirred for added 24 h to at Fe room3O4 solution temperature. The resulting(10 mL) and conjugate then Rh 110 (MIONs-R solution (2.8 110) mL) was was puriﬁed added and by extensivethe mixture dialysis was stirred against for 24 distilled h at room water for threetemperature. nights. AsThe a resulting control, conjug a dyeate solution (MIONs-R with 110) same was molar purified concentration by extensive was dialysis also dialyzedagainst for threedistilled nights. water for three nights. As a control, a dye solution with same molar concentration was also dialyzed for three nights. 3. Results and Discussion 3. Results and Discussion 3.1. Synthesis and Characterization of PTMP-PMAA 3.1. Synthesis and Characterization of PTMP-PMAA Multi-functional water-soluble polymer ligand PTMP-PMAA was synthesized using pentaerythritol Multi-functional water-soluble polymer ligand PTMP-PMAA was synthesized using tetrakis 3-mercaptopropionate as chain transfer agent by free radical polymerization of monomer pentaerythritol tetrakis 3-mercaptopropionate as chain transfer agent by free radical polymerization methacrylicof monomer acid (MAA)methacrylic as described acid (MAA) previously as describe (Schemed previously1). GPC (Scheme elution 1). curve GPC ofelution polymer curve is shownof in Figurepolymer1 and is itsshown molecular in Figure weights 1 and its are molecular given in weights Table1 are. given in Table 1.

Figure 1. GPC (gel permeation chromatography) curve of the PTMP-PMAA obtained with 2% Figure 1. GPC (gel permeation chromatography) curve of the PTMP-PMAA obtained with 2% pentaerythritol tetrakis 3-mercaptopropionate (PTMP). pentaerythritol tetrakis 3-mercaptopropionate (PTMP). Table 1. Molecular weight of 2% PTMP-PMAA. Table 1. Molecular weight of 2% PTMP-PMAA. MAA/PTMP Molecular Weights (g/mol) Sample Yield (%) (mol/mol)MAA/PTMP MolecularMn weightsMwPDI (g/mol) Sample Yield (%) 2% PTMP-PMAA 100/2(mol/mol) M 5,850n Mw 7,420 PDI 1.2 82 2% PTMP-PMAA 100/2 5,850 7,420 1.2 82

Polymers 2016, 8, 392 7 of 16

1 H-NMR spectra were recorded on a 400 MHz Bruker AV400 spectrometer using d6-DMSO as a solvent in a 5 mm quartz NMR tube at room temperature using the δ scale. The 1H-NMR spectra were consistent to the previous works [28,29]. The molecular weights of the polymer ligands were calculated based on the ratio of monomer units attached to the terminal group in the 1H-NMR spectra and compared with molecular weights determined by GPC (data not given). Chemical structure of the polymer was conﬁrmed by 1H-NMR spectroscopy and data as given were consistent with our previous work [28,29]. PTMP-PMAA (d6-DMSO) δ (ppm): δ0.81~1.14; δ1.42~1.54; δ1.61~1.28; δ2.24~2.40; δ2.51~2.84; δ3.17~3.50; δ3.91~4.40. The as synthesized polymer was soluble in EtOH, MeOH, H2O and DMSO. In order to determine the molecular weight of polymer ligand by GPC it was transferred to the THF by converting it into methyl ester using TMS-Diazomethane reagent according to the previous work [30,31]. GPC was performed with an Agilent 1100 instrument using refractive index detector (RID) and THF was used as eluent at a ﬂow rate of 1.0 mL/min at 23 ◦C. The calculated molecular weights were based on a calibration curve for polystyrene standards of narrow polydispersity (Polymer Laboratories). As expected, the molecular weight of the polymer was decreased with the increase in concentration of chain transfer agent.

3.2. Synthesis and Characterization of Polymer Stabilized MIONs MIONs were synthesized by co-precipitation of aqueous iron precursor solution containing Fe3+ and Fe2+ (molar ratio 2:1) by ammonia in the presence of PTMP-PMAA. The size, shape and magnetic properties of the MIONs were controlled by using different molecular weight and concentration of polymer ligand, during their preparation. This polymer ligand has already been proven to be a good capping ligand for the synthesis of cobalt and gold NPs by some of us [21,28,29]. It is known that rapid injection of precursors results in super saturation of chemical species in the reaction mixture which leads to an initial burst of nucleation at once followed by the growth of nuclei leading to the formation of monodisperse inorganic NPs, whereas slow drop-wise addition of precursors results in continuous nucleation and growth process in parallel resulting in broad size distribution of NPs [32]. However, in the case of magnetite NPs, the process is more complicated and it is difﬁcult to control the nucleation and growth processes because iron precursors are initially hydrolyzed in alkaline conditions and then are condensed into iron oxide. Therefore, we dissolved iron precursors in concentrated HCl (38%) in order to avoid their hydrolysis and condensation prior to the addition of precipitating agents. Both iron precursor’s solution and ammonia were rapidly added into the boiling aqueous solution of polymer under nitrogen atmosphere with vigorous stirring in order to keep nucleation and growth processes separate. Rapid injection of precursor is widely used for uniform inorganic NPs synthesis in organic phase by thermal decomposition method [32,33]. The preparation of ultra-small and uniform magnetic NPs with high crystallinity and magnetization through usual co-precipitation method at room temperature is a tedious job, therefore we have carried out co-precipitation process at high temperature (100 ◦C) [20], which increases the reaction rate due to increased diffusion of active species resulting in the formation of NPs with a narrow size distribution [20]. Polymer ligand (PTMP-PMAA) molecules cap the newly formed NPs through interaction between carboxylic acids groups and the iron atoms on NPs surface and thus restrict their further growth by compensating the surface energy due to electrostatic and steric stabilization which yields uniform as well as highly stable NPs. We have already proved this hypothesis by taking samples at different time intervals showing little further growth of NPs with the passage of time [20]. It was also noticed that the use of different ferrous precursor (FeCl2·4H2O instead of FeSO4·7H2O) had no effect on shape, size and size distribution of the MIONs. Moreover, in comparison to the thermal decomposition method, this method involves no use of expensive and toxic organic precursors or solvents but reduces the reaction time and temperature; therefore, it is more economical and eco-friendly. Polymers 2016, 8, 392 8 of 16

Polymers 2016, 8, 392 8 of 16 3.2.1. Effect of Polymer Concentration on Size of MIONs 3.2.1. Effect of Polymer Concentration on Size of MIONs As described earlier the purpose of this research work was to improve the magnetic properties of our previousAs described reported earlier MIONs the purpose prepared of this with research PTMP-PMAA work was[ 20to ].improve In that the report, magnetic MIONs properties prepared withof our 0.768 previous mM concentration reported MIONs of PTMP-PMAAprepared with PTMP-PMAA had hydrodynamic [20]. In diameterthat report, <10 MIONs nm determined prepared bywith DLS 0.768 and mM core concentration size of 4.5 ± of0.4 PTMP-PMAA nm as determined had hydrodynamic by TEM. Those diameter MIONs <10 nm were determined highly water by solubleDLS and but theircore size small of size4.5 ± and 0.4 lowernm as magnetizationdetermined by (45TEM. emu Those·g−1) MIONs restricted were their highly applications water soluble such as magneticbut their separation. small size Therefore, and lower herein, magnetization we gradually (45 emu·g decreased−1) restricted the concentration their applications of PTMP-PMAA such as frommagnetic 0.768 mM separation. all the wayTherefore, down herein, to zero we during gradually preparation decreased of MIONsthe concentration and optimized of PTMP-PMAA the polymer concentrationfrom 0.768 mM to obtain all the MIONs way down with to higher zero during saturation preparation magnetization of MIONs without and optimized compromising the polymer their size, concentration to obtain MIONs with higher saturation magnetization without compromising their dispersibility and stability. size, dispersibility and stability. Figure2a shows the DLS curves of the MIONs prepared with different PTMP-PMAA Figure 2a shows the DLS curves of the MIONs prepared with different PTMP-PMAA concentrations and it was noticed that the hydrodynamic diameters of the MIONs prepared with concentrations and it was noticed that the hydrodynamic diameters of the MIONs prepared with polymer concentrations as low as 0.072 mM did not result in the increase in the size of the MIONs. polymer concentrations as low as 0.072 mM did not result in the increase in the size of the MIONs. However,However, when when the the polymer polymer concentration concentration was was decreased decreased beyond beyond 0.072 0.072 mM, mM, the sizethe ofsize the of MIONs the noticeablyMIONs noticeably increased. increased. Figure2b Figure represent 2b therepresen DLStcurves the DLS of curves the MIONs of the prepared MIONs prepared with 0.072 with and 0.7680.072 mM and PTMP-PMAA 0.768 mM PTMP-PMAA representing representing their hydrodynamic their hydrodynamic diameters todiameters be 9 and to 10 be nm 9 and respectively, 10 nm whichrespectively, strongly which indicates strongly that the indicates a ten times that the decrease a ten times in polymer decrease concentration in polymer didconcentration not much did affect thenot size much of the affect MIONs. the size Therefore, of the MIONs. 0.072 mMTheref wasore, considered 0.072 mM towas be considered the lowest to effective be the lowest polymer ligandeffective concentration polymer andligand thus concentration the MIONs preparedand thus with the 0.072MIONs mM prepared PTMP-PMAA with were0.072 selectedmM forPTMP-PMAA further characterization were selected through for further TEM characterization analysis and the through results TEM were analysis compared and withthe results those of 0.768were mM compared PTMP-PMAA. with those of 0.768 mM PTMP-PMAA.

(a) (b)

FigureFigure 2. 2. (a(a)) DynamicDynamic lightlight scatteringscattering (DLS) curves curves of of the the MIONs MIONs prepared prepared with with different different concentrations of PTMP-PMAA all lower than 0.768 mM; and (b) DLS curves of the MIONs prepared concentrations of PTMP-PMAA all lower than 0.768 mM; and (b) DLS curves of the MIONs prepared with 0.768 and 0.072 mM concentration of PTMP-PMAA. with 0.768 and 0.072 mM concentration of PTMP-PMAA. In order to determine the actual iron oxide core size of these MIONs, we performed the TEM analysisIn order of the to determine MIONs prepared the actual with iron 0.072 oxide and core0.768 size mM of concentrations these MIONs, of we PTMP-PMAA. performed theIt was TEM analysisinteresting of the to MIONs note that prepared the actual with size 0.072 of andthe 0.768MIONs mM was concentrations almost half of of their PTMP-PMAA. hydrodynamic It was interestingdiameters to suggesting note that the the actual presence size ofof theextended MIONs polymer was almost shell half around of their these hydrodynamic nanoparticles diameters when suggestingdispersed thein aqueous presence solutions. of extended The TEM polymer size of shellthe MIONs around prepared these nanoparticles with 0.768 mM when PTMP-PMAA dispersed inwas aqueous found solutions. to be 4.6 nm The with TEM a narrow size of size the distribution MIONs prepared of 0.4 nm with (Figure 0.768 3a) mMand these PTMP-PMAA results were was foundin complete to be 4.6 agreement nm with awith narrow the previous size distribution report [20]. of 0.4However, nm (Figure the TEM3a) and analysis these of results the MIONs were in completeprepared agreement with 0.072 with mM the PTMP-PMAA previous report revealed [20]. that However, their size the was TEM 4.8 analysis nm with of a thesize MIONs distribution prepared of with0.6 0.072nm (Figure mM PTMP-PMAA 3b), which is revealed very much that theircomparable size was to 4.8the nm MIONs with aprepared size distribution with 0.768 of 0.6mM nm (FigurePTMP-PMAA.3b), which The is veryslight much increase comparable in size and to size the distribution MIONs prepared of these with MIONs 0.768 in mMcomparison PTMP-PMAA. to the Theten slight times increase decrease in in size the and polymer size distribution concentration of thesecan be MIONs considered in comparison as insignificant to the and ten legitimate. times decrease in the polymer concentration can be considered as insigniﬁcant and legitimate.

Polymers 2016, 8, 392 9 of 16 Polymers 2016, 8, 392 9 of 16

Polymers 2016, 8, 392 9 of 16

(a) (b)

Figure 3. TEM image (anda) histogram (insert) of MIONs prepared with: (a) 0.768(b) mM PTMP-PMAA; Figure 3. TEM image and histogram (insert) of MIONs prepared with: (a) 0.768 mM PTMP-PMAA; and (b) 0.072 mM PTMP-PMAA. andFigure (b) 0.072 3. TEM mM image PTMP-PMAA. and histogram (insert) of MIONs prepared with: (a) 0.768 mM PTMP-PMAA; and (b) 0.072 mM PTMP-PMAA. Conversely, the MIONs obtained without PTMP-PMAA had TEM diameter more than 10 nm Conversely, the MIONs obtained without PTMP-PMAA had TEM diameter more than 10 nm with Conversely,very large sizethe MIONsdistribution obtained range without 2.9 nm PTMP-PMAA (Figure 4) and had were TEM notdiameter very wellmore dispersed than 10 nm in with very large size distribution range 2.9 nm (Figure4) and were not very well dispersed in aqueous withaqueous very solution large size as they distribution tended agglomerate range 2.9 nm and (Figure settle down4) and under were force not veryof gravity well withindispersed a few in solution as they tended agglomerate and settle down under force of gravity within a few minutes, aqueousminutes, solutionperhaps asdue they to tendedtheir large agglomerate size, high and surf settleace energy, down underetc. These force results of gravity revealed within that a fewthe perhapsminutes,concentration due perhaps to theirof the due large PTMP-PMAA to size,their high large surface(carbonyl size, high energy, groups surf etc.ace to Theseenergy, iron precursor results etc. These revealed ratio) results played that revealed the an concentration important that the ofconcentrationrole, the PTMP-PMAA and is a ofcritical the (carbonyl PTMP-PMAA factor in groups determining (carbonyl to iron precursorsize groups and size-distributionto ratio) iron played precursor an importantof ratio) the MIONs.played role, an andIt importantshould is a critical be factorrole,noted inand that determining is the a DLScritical diameter size factor and of size-distributionin thedetermining bare MIONs size of was the and MIONs.observed size-distribution It to should be more beof than notedthe 70MIONs. that nm thebecause It DLS should of diameter their be ofnotedhigh the bareagglomeration, that MIONs the DLS was diameter which observed was of the further to bare be more MIONsconfirmed than was 70 by observed nm TEM because (Figure to be of 4).more their than high 70 agglomeration, nm because of their which washigh further agglomeration, conﬁrmed which by TEM was (Figure further4 ).confirmed by TEM (Figure 4).

Figure 4. TEM image of Bare MIONs with histogram (inset). Figure 4. TEM image of Bare MIONs with histogram (inset). In conclusion, it wasFigure found 4. TEM that image the MIONs of Bare were MIONs successfully with histogram synthesized (inset). with small size (4.8 ± 0.6 nm)In conclusion, using 0.072 itmM was PTMP-PMAA found that the which MIONs is almost were successfully ten times lower synthesized concentration with small as compared size (4.8 to ± 0.60.768 Innm) conclusion,mM using used 0.072 previously itmM was PTMP-PMAA found without that significant which the MIONs is almost increase were ten successfullytimesin the lower size concentrationand synthesized size distribution as with compared small of the to size (4.80.768MIONs.± 0.6 mM nmFurthermore, used) using previously 0.072 it would mM without also PTMP-PMAA improve significant the which maincreasegnetic is almostinproperties the size ten of timesand the sizeMIONs lower distribution by concentration lowering of the as comparedMIONs.polymer Furthermore,tocontents 0.768 mM percentage. usedit would previously also improve without the signiﬁcant magnetic increaseproperties in of the the size MIONs and size by lowering distribution the of thepolymer MIONs. contents Furthermore, percentage. it would also improve the magnetic properties of the MIONs by lowering the polymer contents percentage.

Polymers 2016, 8, 392 10 of 16

3.2.2. CharacterizationPolymers 2016, 8, 392 of MIONs 10 of 16

Further3.2.2. Characterization characterization of MIONs of the MIONs was performed by taking FTIR, TGA, XRD and VSM analysis of theFurther MIONs characterization prepared withof the 0.072 MIONs and was 0.768 performed mM PTMP-PMAA, by taking FTIR, and TGA, results XRD and were VSM compared. In orderanalysis to of know the MIONs the surface prepared chemistry with 0.072 of MIONs, and 0.768 they mM were PTMP-PMAA, characterized and by results Fourier were transform infraredcompared. spectroscopy (Figure5). In FTIR spectrum of polymer ligand, a strong absorption peak at 1704 cm−1 correspondsIn order to know to the the surface asymmetric chemistry stretching of MIONs, they of carbonyl were characterized (–CO–) groupby Fourier of polymertransform ligand, which isinfrared extensively spectroscopy reduced (Figure in 5). spectrum In FTIR spectrum of MIONs of polymer with polymer ligand, a strong indicating absorption the attachmentpeak at to 1704 cm−1 corresponds to the asymmetric stretching of carbonyl (–CO–) group of polymer ligand, the surface of iron oxide NPs [34]. Additionally, a broad peak at 3124 cm−1 is attributed to the which is extensively reduced in spectrum of MIONs with polymer indicating the attachment to the O–H stretchingsurface of ofiron carboxylic oxide NPs acid[34]. Additionally, groups and a isbroad observed peak at in3124 FTIR cm−1 spectra is attributed of PTMP-PMAA to the O–H and MIONs@PTMP-PMAAstretching of carboxylic [28,35 acid]. Furthermore, groups and is the observed asymmetric in FTIR and spectra symmetric of PTMP-PMAA stretching and bands of carboxylateMIONs@PTMP-PMAA (–COO–) present [28,35]. in pure Furthermore, polymer ligand the asy spectrummmetric and are shiftedsymmetric from stretching 1485, 1390bands to of 1556 and 1402 cmcarboxylate−1, respectively, (–COO–) in present the spectrum in pure polymer of MIONs ligand protected spectrum withare shifted polymer from ligand,1485, 1390 which to 1556 conﬁrms −1 the presenceand 1402 of polymercm , respectively, ligand on in NPs the surface.spectrum Finally,of MIONs the protected characteristic with polymer peaks appearing ligand, which at 592 and confirms the presence of polymer ligand on NPs surface. Finally, the characteristic peaks appearing 448 cm−1 are assigned to the torsion vibration and stretching mode of Fe–O bond of magnetite present at 592 and 448 cm−1 are assigned to the torsion vibration and stretching mode of Fe–O bond of only inmagnetite FTIR spectra present of only MIONs in FTIR with spectr PTMP-PMAAa of MIONs with [35 ,PTMP-PMAA36]. [35,36].

FigureFigure 5. Fourier5. Fourier transform transform infraredinfrared spectroscopyspectroscopy (FTIR) (FTIR) spectra spectra of PTMP-PMAA of PTMP-PMAA and and MIONs@PTMP-PMAA. MIONs@PTMP-PMAA. The crystalline structure of the MIONs was determined by XRD measurement as shown in TheFigure crystalline 6. The peaks structure present of at the2θ =MIONs 30.1°, 35.5°, was 43.3°, determined 53.4°, 57.3° byandXRD 62.7° corresponding measurement to as(220), shown in Figure6(311),. The (400), peaks (422), present (511) at and 2 θ =(440) 30.1 reflections◦, 35.5◦, 43.3of magnetite,◦, 53.4◦, 57.3 respectively,◦ and 62.7 can◦ corresponding be clearly seen, to (220), indicating the presence of crystalline spinel structured magnetite (Fe3O4) phase of iron oxide [37]. (311), (400), (422), (511) and (440) reﬂections of magnetite, respectively, can be clearly seen, indicating However, the presence of Fe2O3 is not completely ruled out as the phase of the iron oxide in the presencemagnetic of NPs crystalline cannot be spinel confirmed structured from simple magnetite FTIR and (FeXRD3O as4 )they phase are less of ironsensitive oxide to them [37]. [20]. However, the presence of Fe2O3 is not completely ruled out as the phase of the iron oxide in magnetic NPs cannot be conﬁrmed from simple FTIR and XRD as they are less sensitive to them [20]. Polymers 2016, 8, 392 11 of 16

Figure 6. X-ray powder diffraction (XRD) spectrum of MIONs prepared with PTMP-PMAA. Figure 6. X-ray powder diffraction (XRD) spectrum of MIONs prepared with PTMP-PMAA. To further investigate the thermal stability of the MIONs, and to deduce the iron oxide and polymer contents of the NPs, thermal gravimetric analysis was performed for PTMP-PMAA, MIONs prepared with 0.072 and 0.768 mM PTMP-PMAA as well as for bare MIONs prepare without the use of any polymer ligand (Figure 7). It can be seen that neat polymer ligand (PTMP-PMAA) was almost completely decomposed at 480 °C with almost no residue left. The magnetic iron oxide and polymer contents of the MIONs prepared with 0.768 mM PTMP-PMAA were approximately 33% and 67%, respectively. However, the iron oxide and polymer contents of the MIONs prepared with 0.072 mM PTMP-PMAA were 42% and 58%, respectively, as expected. Therefore, from TGA analysis, it was concluded that the magnetic iron oxide wt % in the MIONs with PTMP-PMAA (0.072 mM) was higher than those prepared with 0.768 mM PTMP-PMAA which contributed towards the higher saturation magnetization of the MIONs in this case.

Figure 7. Thermogravimetric analysis (TGA) curves of PTMP-PMAA, MIONs with PTMP-PMAA (0.072 mM and 0.768 mM) and bare MIONs.

Saturation magnetization of MIONs prepared with 0.072 and 0.768 mM PTMP-PMAA ligands was determined by Vibrating Sample Magnetometer (VSM) at room temperature. In order to calculate the exact magnetization of these MIONs, the effect of magnetically dead polymer layer was excluded and then compared with magnetization of bare MIONs without polymer (Figure 8). The corrected saturation magnetization of the MIONs prepared with 0.768 mM PTMP-PMAA, having TEM diameter about 4.6 nm, was found to be 45 emu·g−1, which is comparable to the literature for MIONs of comparable dimensions [20]. The low saturation magnetization of 4.6 nm MIONs

Polymers 2016, 8, 392 11 of 16

Figure 6. X-ray powder diffraction (XRD) spectrum of MIONs prepared with PTMP-PMAA. Polymers 2016, 8, 392 11 of 16

To further investigate the thermal stability of the MIONs, and to deduce the iron oxide and polymer Tocontents further of investigate the NPs, thermal the thermal gravimetri stabilityc ofanalysis the MIONs, was performed and to deduce for PTMP-PMAA, the iron oxide andMIONs preparedpolymer with contents 0.072 and of the 0.768 NPs, mM thermal PTMP-PMAA gravimetric as analysis well as was for performed bare MIONs for PTMP-PMAA, prepare without MIONs the use of anyprepared polymer with ligand 0.072 (Figure and 0.768 7). mM It can PTMP-PMAA be seen that as neat well aspolymer for bare ligand MIONs (PTMP-PMAA) prepare without was the use almost of any polymer ligand (Figure7). It can be seen that neat polymer ligand (PTMP-PMAA) was almost completely decomposed at 480 °C with almost no residue left. The magnetic iron oxide and polymer completely decomposed at 480 ◦C with almost no residue left. The magnetic iron oxide and polymer contents of the MIONs prepared with 0.768 mM PTMP-PMAA were approximately 33% and 67%, contents of the MIONs prepared with 0.768 mM PTMP-PMAA were approximately 33% and 67%, respectively.respectively. However, However, the the iron iron oxide oxide and and polymer polymer contentscontents of of the the MIONs MIONs prepared prepared with with 0.072 0.072 mM mM PTMP-PMAAPTMP-PMAA were were 42% 42% and and 58%, 58%, respectively, respectively, as expected.expected. Therefore, Therefore, from from TGA TGA analysis, analysis, it was it was concludedconcluded that that the the magnetic magnetic iron oxideoxide wt wt % in% thein MIONsthe MIONs with PTMP-PMAAwith PTMP-PMAA (0.072 mM) (0.072 was highermM) was higherthan than those those prepared prepared with 0.768with mM 0.768 PTMP-PMAA mM PTMP-PMAA which contributed which contributed towards the highertowards saturation the higher saturationmagnetization magnetization of the MIONs of the in MIONs this case. in this case.

FigureFigure 7. Thermogravimetric 7. Thermogravimetric analysis analysis (TGA) (TGA) curves curves ofof PTMP-PMAA,PTMP-PMAA, MIONs MIONs with with PTMP-PMAA PTMP-PMAA (0.072(0.072 mM mM and and 0.768 0.768 mM) mM) and and bare bare MIONs. MIONs.

SaturationSaturation magnetization magnetization of ofMIONs MIONs prepared prepared with 0.0720.072 and and 0.768 0.768 mM mM PTMP-PMAA PTMP-PMAA ligands ligands was wasdetermined determined by by Vibrating Vibrating SampleSample Magnetometer Magnetomet (VSM)er (VSM) at room at temperature. room temperature. In order to calculateIn order to calculatethe exact the exact magnetization magnetization of these of MIONs, these MIONs, the effect the of magnetically effect of magn deadetically polymer dead layer polymer was excluded layer was excludedand then and compared then compared with magnetization with magnetization of bare MIONs of bare without MIONs polymer without (Figure polymer8). The (Figure corrected 8). The correctedsaturation saturation magnetization magnetization of the MIONs of the prepared MIONs with prepared 0.768 mM with PTMP-PMAA, 0.768 mM having PTMP-PMAA, TEM diameter having about 4.6 nm, was found to be 45 emu·g−1, which is comparable to the literature for MIONs of TEM diameter about 4.6 nm, was found to be 45 emu·g−1, which is comparable to the literature for comparable dimensions [20]. The low saturation magnetization of 4.6 nm MIONs compared to the bare MIONs of comparable dimensions [20]. The low saturation magnetization of 4.6 nm MIONs MIONs is due to their ultra-small size as saturation magnetization of magnetic NPs is directly related

to their size [38]. However, it was interesting to note that the saturation magnetization of our MIONs prepared with 0.072 mM PTMP-PMAA with TEM diameter 4.8 nm was found to be 58 emu·g−1, which is much higher than that of MIONs prepared with 0.768 mM. We attribute this increase in the saturation magnetization of the MIONs to the decrease in magnetically dead polymer contents of the MIONs as mentioned earlier. Polymers 2016, 8, 392 12 of 16 Polymers 2016, 8, 392 12 of 16 compared to the bare MIONs is due to their ultra-small size as saturation magnetization of magnetic compared to the bare MIONs is due to their ultra-small size as saturation magnetization of magnetic NPs is directly related to their size [38]. However, it was interesting to note that the saturation NPs is directly related to their size [38]. However, it was interesting to note that the saturation magnetization of our MIONs prepared with 0.072 mM PTMP-PMAA with TEM diameter 4.8 nm magnetization of our MIONs prepared with 0.072 mM PTMP-PMAA with TEM diameter 4.8 nm was found to be 58 emu·g−1, which is much higher than that of MIONs prepared with 0.768 mM. We was found to be 58 emu·g−1, which is much higher than that of MIONs prepared with 0.768 mM. We attribute this increase in the saturation magnetization of the MIONs to the decrease in magnetically Polymersattribute2016 this, 8, increase 392 in the saturation magnetization of the MIONs to the decrease in magnetically12 of 16 dead polymer contents of the MIONs as mentioned earlier. dead polymer contents of the MIONs as mentioned earlier.

Figure 8. Magnetization curves of MIONs prepared with 0.072, 0.768 and 0.00 mM concentration of Figure 8. MagnetizationMagnetization curves curves of of MIONs MIONs prepared prepared with with 0.072, 0.072, 0.768 0.768 and and 0.00 0.00 mM mM concentration concentration of PTMP-PMAA. PTMP-PMAA.of PTMP-PMAA. However, the magnetization of the MIONs was still much lower than that of bare MIONs However, thethe magnetization magnetization of of the the MIONs MIONs was was still muchstill much lower lower than that than of barethat MIONsof bare prepared MIONs prepared without polymer ligand (Figure 8). Therefore, it can be concluded that water-soluble withoutprepared polymer without ligand polymer (Figure ligand8). Therefore,(Figure 8). it Ther canefore, be concluded it can be that concluded water-soluble that water-soluble MIONs with MIONs with comparable size were prepared with ten times less polymer concentration as compared MIONscomparable with sizecomparable were prepared size were with prepared ten times with less ten polymer times less concentration polymer concentration as compared as to compared previous to previous report [20], which resulted in a great increase in the magnetization of the MIONs. toreport previous [20], whichreport resulted[20], which in a resulted great increase in a great in the increase magnetization in the magnetization of the MIONs. of the MIONs. It is interesting to note that the increase in the saturation magnetization of the MIONs made It is interesting to note that the increaseincrease inin thethe saturationsaturation magnetization of the MIONs made them able to be collected with the help of an external magnet from their aqueous solution without them able to be collected with the help of an externalexternal magnetmagnet from theirtheir aqueousaqueous solution without compromising their stability and dispersibility, as shown in the Figure 9. We believe that this would compromising their stability and dispersibility, as shownshown in the Figure9 9.. WeWe believebelieve thatthat thisthis wouldwould increase the scope of applications of these MIONs such as the magnetic separations of biomolecules increase the scope of applications of these MIONs such as the magnetic separations of biomolecules like proteins, nucleic acids and even cells. like proteins, nucleic acids and even cells.cells.

Figure 9. Aqueous dispersion of the MIONs prepared with: 0.768 mM (left); and 0.072 mM (right) Figure 9. Aqueous dispersion of the MIONs prepared with: 0.768 mM (left); and 0.072 mM (right) Figureconcentrations 9. Aqueous of PTMP-PMAA dispersion of under the MIONs the effect prepared of external with: magnetic 0.768 mM (1.5 ( leftT) after); and 1 h. 0.072 mM (right) concentrations of PTMP-PMAA under the effect of external magnetic (1.5 T)T) afterafter 11 h.h. Dried MIONs prepared with PTMP-PMAA were highly stable and readily redispersible in Dried MIONs prepared with PTMP-PMAA were highly stable and readily redispersible in water,Dried pH and MIONs salt stability prepared tests with were PTMP-PMAA performed with were aqueous highly dispersions stable and of readily MIONs redispersible prepared with in water, pH and salt stability tests were performed with aqueous dispersions of MIONs prepared with water, pH and salt stability tests were performed with aqueous dispersions of MIONs prepared with PTMP-PMAA (0.072 mM) and were found to be stable within a wide range of pH (5–10) and high concentrations (1 M) of salt (NaCl). High stability of the MIONs dispersions can be attributed to large negative zeta potential (−49 mV) and excellent electrostatic as well as steric stabilization by polymer ligand owing to the large abundance of carboxylic acid groups [25]. Polymers 2016, 8, 392 13 of 16

PTMP-PMAA (0.072 mM) and were found to be stable within a wide range of pH (5–10) and high concentrations (1 M) of salt (NaCl). High stability of the MIONs dispersions can be attributed to largePolymers negative2016, 8, 392 zeta potential (−49 mV) and excellent electrostatic as well as steric stabilization13 of by 16 polymer ligand owing to the large abundance of carboxylic acid groups [25].

3.2.3. Toxicity Toxicity Analysis of MIONs Biocompatibility ofof the the MIONs@PTMP-PMAA MIONs@PTMP-PMAA was determinedwas determined through through their in vitrotheircytotoxicity in vitro cytotoxicityanalysis performed analysis onperformed HepG2 cellson HepG2 as demonstrated cells as demonstrated in experimental in experimental section. Figure section. 10 showsFigure the10 showspercent the cell percent viability cell of HepG2 viability cells, of whichHepG2 was cells, determined which was by MTT-assay,determined after by incubationMTT-assay, of after cells incubationwith different of cells concentrations with different of MIONs concentratio preparedns of with MIONs 0.072 prepared mM PTMP-PMAA with 0.072 mM for 24, PTMP-PMAA 48 and 72 h. forCell 24, viability 48 and 72 determined h. Cell viability at zero determined concentration at zero of MIONsconcentration or polymer of MIONs ligand or polymer was taken ligand as 100%. was takenThese asin 100%. vitro cytotoxicityThese in vitro tests cytotoxicity clearly demonstrate tests clearly demonstrate that MIONs@PTMP-PMAA that MIONs@PTMP-PMAA were completely were completelynon-toxic and non-toxic cells survived and cells well survived even at higher well even concentrations at higher (500concentrationsµg·mL−1) due(500 to µg·mL nontoxic−1) due nature to nontoxicof PTMP-PMAA nature of [PTMP-PMAA5], however, cell [5], viabilityhowever, more cell viability than 100% more in than few 100% cases in was few maybe cases was due maybe to cell dueproliferation. to cell proliferation. In opposite In to opposite that, bare to MIONs that, bare have MIONs time and have dose time dependent and dose high dependent toxicity high as described toxicity asby described us previously by us [ 25previously], which was[25], attributedwhich was to attrib theuted known to the fact known that bare fact MIONs that bare act MIONs as a source act as ofa sourceferrous of ions ferrous and ions highly and reactive highly reactive hydroxyl hydroxyl radicals radicals causing causing oxidative oxidative and free and radical free radical stress tostress the tocells the [ 39cells]. [39].

Figure 10. Cell viability of HepG2 determined by by MTT-a MTT-assayssay after 24, 48 and 72 h of incubation with various concentrations of MIONs@PTMP-PMAA.

3.3. Functionalization Functionalization of MIONs with Fluorescent Dye ItIt was determined through characterizationcharacterization that MIONs@PTMP-PMAA had had high water dispersibility andand excellent excellent stability stability in a widein a rangewide of range pH and of salt pH concentration and salt andconcentration multifunctional and multifunctionalsurface chemistry, surface due chemistry, to polymer due shell to polymer around them,shell around which them, make which them highlymake them suitable highly for suitablebio-applications for bio-applications such as contrast such as agents contrast for MRI,agents magnetic for MRI, separationmagnetic separation of biomolecules of biomolecules and cells, and developmentcells, and development of NPs based of NPs drug based delivery drug systems. delivery Furthermore, systems. Furthermore, the presence the of abundantpresence of abundantcarboxylic of acid carboxylic (–COOH) acid and (–COOH) thiol (–SH) and groups thiol (– onSH) MIONs groups surface on MIONs also provide surface greatalso provide opportunities great opportunitiesto simultaneously to simultaneously modify these NPsmodify with these targeting, NPs wi tracingth targeting, and therapeutic tracing and agents therapeutic for multipurpose agents for multipurposebio-probes. We bio-probes. demonstrated We demonstrated the functionalization the func oftionalization these MIONs of withthese a MIONs fluorescent withdye a fluorescent molecule, dyeRhodamine molecule, 110 Rhodamine (Rh 110) bearing 110 (Rh primary 110) bearing amine group primary through amine EDC/NHS group through coupling EDC/NHS reaction, coupling which is reaction,the easiest which and most is the feasible easiest reactionand most of feasible linking re moleculesaction of throughlinking molecules primary amine through and primary carboxylic amine acid andgroups carboxylic by amide acid bond groups formation. by amide bond formation. MIONs werewere conjugated conjugated with with Rh Rh 110 110 by following by follow theing procedure the procedure described described in Section in 2.6 Section. Aqueous 2.6. Aqueoussolution ofsolution MIONs-Rh of MIONs-Rh 110 conjugates, 110 conjugates, after extensive after dialysis extensive for three dialysis nights for against three nights distilled against water, distilledshowed fluorescewater, showed under fluoresce UV light asunder shown UV in light the Figureas shown 11. Asin athe control, Figure a 11. dye As solution a control, with a same dye solutionmolar concentration with same molar was also concentration dialyzed for was three also nights. dialyzed It was for noted three that nights. the control It was experiment noted that with the equivalent concentration of Rh 110 did not exhibit any fluorescence after dialysis, which was the indication of attachment of Rh 110 with MIONs. Polymers 2016, 8, 392 14 of 16

Polymerscontrol2016 experiment, 8, 392 with equivalent concentration of Rh 110 did not exhibit any fluorescence after14 of 16 dialysis, which was the indication of attachment of Rh 110 with MIONs.

Figure 11. The UV–vis absorption (black) and fluorescent emission spectra of aqueous solution of Figure 11. The UV–vis absorption (black) and fluorescent emission spectra of aqueous solution of MIONs-Rh 110 conjugate after extensive dialysis, insert shows optical image in ambient light (left), MIONs-Rh 110 conjugate after extensive dialysis, insert shows optical image in ambient light (left), and in UV light (right). and in UV light (right). 4. Conclusions 4. Conclusions Biocompatible ultra-small magnetic iron oxide (Fe3O4) NPs were prepared by co-precipitation ofBiocompatible aqueous iron precursor ultra-small solution magnetic at high iron temper oxideature (Fe3 Oin4 )the NPs presence were prepared of water-soluble by co-precipitation thioether of aqueousend-functionalized iron precursor polymer solution ligand atPTMP-PMAA. high temperature The saturation in the presence magnetization of water-soluble of the MIONs thioether was end-functionalizedfurther improved polymerby optimizing ligand the PTMP-PMAA. polymer ligand The concentration saturation compared magnetization to our of previous the MIONs work. was furtherThe increase improved in bymagnetization optimizing theof the polymer MIONs ligand made concentrationit possible to manipulate compared tothem our with previous external work. Themagnets increase in in their magnetization aqueous solution, of the MIONs which madeincreases it possible their scope to manipulate of applications. them Furthermore, with external it magnets was in theirfound aqueous that these solution, MIONs which were increasesfairly uniform their scopein size of and applications. could be dried Furthermore, by solvent it wasevaporation, found that thesewhich MIONs could were be stored fairly for uniform a long intime. size The and dried could powders be dried of by MIONs solvent were evaporation, readily re-dispersible which could in be storedwater for without a long time.any significant The dried change powders in their of MIONs size and were size readily distribution. re-dispersible MIONs aqueous in water dispersion without any significantwas highly change stable, in theireven size at andhigh sizesalt distribution.concentration MIONs and also aqueous over dispersiona wide range was of highly pH. The stable, concentration of polymer ligand showed significant effect on the size and magnetic properties of even at high salt concentration and also over a wide range of pH. The concentration of polymer ligand MIONs. MIONs were found to be highly biocompatible as determined by MTT-assay. The showed significant effect on the size and magnetic properties of MIONs. MIONs were found to be bio-applicability of MIONs demonstrated by successfully coupling them with fluorescent dye highly biocompatible as determined by MTT-assay. The bio-applicability of MIONs demonstrated by Rhodamine (Rh 110) through formation of amide bond linkage between carboxylic acid (–COOH) successfully coupling them with fluorescent dye Rhodamine (Rh 110) through formation of amide groups of MIONs and primary amine (–NH2) groups of dye. Similarly, rich surface chemistry of bondthese linkage MIONs between stabilized carboxylic with PTMP-PMAA acid (–COOH) can groups be exploited of MIONs for the and simultaneous primary amine conjugation (–NH2) groupsof a of dye.variety Similarly, of therapeutic, rich surface targeting, chemistry and oflabeling these MIONsagents for stabilized several withbio-analytical PTMP-PMAA and drug can be delivery exploited forapplications. the simultaneous conjugation of a variety of therapeutic, targeting, and labeling agents for several bio-analytical and drug delivery applications. Acknowledgments: This work was supported by the Natural Science Foundation of Hubei Province Acknowledgments:(Q20141006), China;This and work Opening was supportedFunding from by theMinistry Natural of Education Science Foundation for Key Materials of Hubei and Province Systems (Q20141006),for Large China;Scale and Battery Opening Laboratory, Funding China. from Ministry of Education for Key Materials and Systems for Large Scale Battery Laboratory,Author Contributions: China. This article is a result of a valuable contribution from all the listed co-authors. Bien Tan Authorencouraged Contributions: the idea Thisof the article study is aand result provided of a valuable valuable contribution advice; Muhammad from all theIrfan listed Majeed co-authors. designed Bien and Tan encouragedconducted the the idea main of theresearch study work; and provided Jiaojiao valuableGuo contri advice;buted Muhammadin compiling Irfanthe results Majeed and designed Wei Yan and helped conducted in thewriting main research this manuscript work; Jiaojiao and in correspondence. Guo contributed in compiling the results and Wei Yan helped in writing this manuscript and in correspondence. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Zhang, L.; Dong, W.F.; Sun, H.B. Multifunctional superparamagnetic iron oxide nanoparticles: Design, synthesis and biomedical photonic applications. Nanoscale 2013, 5, 7664–7684. [CrossRef][PubMed] 2. Colombo, M.; Carregal-Romero, S.; Casula, M.F.; Gutierrez, L.; Morales, M.P.; Bohm, I.B.; Heverhagen, J.T.; Prosperi, D.; Parak, W.J. Biological applications of magnetic nanoparticles. Chem. Soc. Rev. 2012, 41, 4306–4334. [CrossRef][PubMed] Polymers 2016, 8, 392 15 of 16

3. Wei, W.; Zhaohui, W.; Taekyung, Y.; Changzhong, J.; Woo-Sik, K. Recent progress on magnetic iron oxide nanoparticles: Synthesis, surface functional strategies and biomedical applications. Sci. Technol. Adv. Mater. 2015, 16, 023501. 4. Yan, K.; Li, H.; Li, P.; Zhu, H.; Shen, J.; Yi, C.; Wu, S.; Yeung, K.W.K.; Xu, Z.; Xu, H.; et al. Self-assembled magnetic ﬂuorescent polymeric micelles for magnetic resonance and optical imaging. Biomaterials 2014, 35, 344–355. [CrossRef][PubMed] 5. Li, Z.; Yi, P.W.; Sun, Q.; Lei, H.; Zhao, H.L.; Zhu, Z.H.; Smith, S.C.; Lan, M.B.; Lu, G.Q.M. Ultrasmall water-soluble and biocompatible magnetic iron oxide nanoparticles as positive and negative dual contrast agents. Adv. Funct. Mater. 2012, 22, 2387–2393. [CrossRef] 6. Blanco-Andujar, C.; Ortega, D.; Southern, P.; Pankhurst, Q.A.; Thanh, N.T.K. High performance multi-core iron oxide nanoparticles for magnetic hyperthermia: Microwave synthesis, and the role of core-to-core interactions. Nanoscale 2015, 7, 1768–1775. [CrossRef][PubMed] 7. Ramesh, R.; Ponnusamy, S.; Muthamizhchelvan, C. Synthesis, properties and heating characteristics of bovine

serum albumin coated Fe3O4 magnetic ﬂuid for magnetic ﬂuid hyperthermia application. Sci. Adv. Mater. 2013, 5, 1250–1255. [CrossRef] 8. Lu, W.; Ling, M.; Jia, M.; Huang, P.; Li, C.; Yan, B. Facile synthesis and characterization of

polyethylenimine-coated Fe3O4 superparamagnetic nanoparticles for cancer cell separation. Mol. Med. Rep. 2014, 9, 1080–1084. [PubMed] 9. Gao, F.; Qu, H.; Duan, Y.; Wang, J.; Song, X.; Ji, T.; Cao, L.; Nie, G.; Sun, S. Dopamine coating as a general and

facile route to biofunctionalization of superparamagnetic Fe3O4 nanoparticles for magnetic separation of proteins. RSC Adv. 2014, 4, 6657–6663. [CrossRef] 10. Quinto, C.A.; Mohindra, P.; Tong, S.; Bao, G. Multifunctional superparamagnetic iron oxide nanoparticles for combined chemotherapy and hyperthermia cancer treatment. Nanoscale 2015, 7, 12728–12736. [CrossRef] [PubMed] 11. Oka, C.; Ushimaru, K.; Horiishi, N.; Tsuge, T.; Kitamoto, Y. Core–shell composite particles composed of biodegradable polymer particles and magnetic iron oxide nanoparticles for targeted drug delivery. J. Magn. Magn. Mater. 2015, 381, 278–284. [CrossRef] 12. Martin, M.; Salazar, P.; Villalonga, R.; Campuzano, S.; Pingarron, J.M.; Gonzalez-Mora, J.L. Preparation of

core-shell Fe3O4@poly(dopamine) magnetic nanoparticles for biosensor construction. J. Mater. Chem. B 2014, 2, 739–746. [CrossRef] 13. Barrow, M.; Taylor, A.; Murray, P.; Rosseinsky, M.J.; Adams, D.J. Design considerations for the synthesis of polymer coated iron oxide nanoparticles for stem cell labelling and tracking using MRI. Chem. Soc. Rev. 2015, 44, 6733–6748. [CrossRef][PubMed] 14. Palui, G.; Aldeek, F.; Wang, W.; Mattoussi, H. Strategies for interfacing inorganic nanocrystals with biological systems based on polymer-coating. Chem. Soc. Rev. 2015, 44, 193–227. [CrossRef][PubMed] 15. Hood, A.M.; Mari, M.; Muñoz-Espí, R. Synthetic strategies in the preparation of polymer/inorganic hybrid nanoparticles. Materials 2014, 7, 4057–4087. [CrossRef] 16. Xin, H.; Hui, Z.; Liyun, L.; Bien, T. Preparation of nanoparticles with multi-functional water-soluble polymer ligands. Prog. Chem. 2010, 22, 953–961. 17. Ling, D.; Hackett, M.J.; Hyeon, T. Surface ligands in synthesis, modification, assembly and biomedical applications of nanoparticles. Nano Today 2014, 9, 457–477. [CrossRef] 18. Wang, W.; Ji, X.; Na, H.B.; Safi, M.; Smith, A.; Palui, G.; Perez, J.M.; Mattoussi, H. Design of a multi-dopamine-modified polymer ligand optimally suited for interfacing magnetic nanoparticles with biological systems. Langmuir 2014, 30, 6197–6208. [CrossRef][PubMed] 19. Pernia Leal, M.; Rivera-Fernandez, S.; Franco, J.M.; Pozo, D.; de la Fuente, J.M.; Garcia-Martin, M.L. Long-circulating pegylated manganese ferrite nanoparticles for mri-based molecular imaging. Nanoscale 2015, 7, 2050–2059. [CrossRef][PubMed] 20. Li, Z.; Tan, B.; Allix, M.; Cooper, A.I.; Rosseinsky, M.J. Direct coprecipitation route to monodisperse dual-functionalized magnetic iron oxide nanocrystals without size selection. Small 2008, 4, 231–239. [CrossRef][PubMed] 21. Lu, L.T.; Tung, L.D.; Robinson, I.; Ung, D.; Tan, B.; Long, J.; Cooper, A.I.; Fernig, D.G.; Thanh, N.T.K. Size and shape control for water-soluble magnetic cobalt nanoparticles using polymer ligands. J. Mater. Chem. 2008, 18, 2453–2458. [CrossRef] Polymers 2016, 8, 392 16 of 16

22. Razzaque, S.; Hussain, S.; Hussain, I.; Tan, B. Design and utility of metal/metal oxide nanoparticles mediated by thioether end-functionalized polymeric ligands. Polymers 2016, 8, 156. [CrossRef] 23. Oanh, V.T.K.; Lam, T.D.; Thu, V.T.; Lu, L.T.; Nam, P.H.; Tam, L.T.; Manh, D.H.; Phuc, N.X. A novel route for

preparing highly stable Fe3O4 ﬂuid with poly(acrylic acid) as phase transfer ligand. J. Electron. Mater. 2016, 45, 4010–4017. [CrossRef] 24. Davis, K.; Qi, B.; Witmer, M.; Kitchens, C.L.; Powell, B.A.; Mefford, O.T. Quantitative measurement of ligand exchange on iron oxides via radiolabeled oleic acid. Langmuir 2014, 30, 10918–10925. [CrossRef][PubMed] 25. Majeed, M.I.; Lu, Q.; Yan, W.; Li, Z.; Hussain, I.; Tahir, M.N.; Tremel, W.; Tan, B. Highly water-soluble

magnetic iron oxide (Fe3O4) nanoparticles for drug delivery: Enhanced in vitro therapeutic efficacy of doxorubicin and mion conjugates. J. Mater. Chem. B 2013, 1, 2874–2884. [CrossRef] 26. Wang, H.; Shen, J.; Li, Y.; Wei, Z.; Cao, G.; Gai, Z.; Hong, K.; Banerjee, P.; Zhou, S. Magnetic iron oxide-fluorescent carbon dots integrated nanoparticles for dual-modal imaging, near-infrared light-responsive drug carrier and photothermal therapy. Biomater. Sci. 2014, 2, 915–923. [CrossRef] 27. Shi, D.; Sadat, M.E.; Dunn, A.W.; Mast, D.B. Photo-fluorescent and magnetic properties of iron oxide nanoparticles for biomedical applications. Nanoscale 2015, 7, 8209–8232. [CrossRef][PubMed] 28. Hussain, I.; Graham, S.; Wang, Z.X.; Tan, B.; Sherrington, D.C.; Rannard, S.P.; Cooper, A.I.; Brust, M. Size-controlled synthesis of near-monodisperse gold nanoparticles in the 1–4 nm range using polymeric stabilizers. J. Am. Chem. Soc. 2005, 127, 16398–16399. [CrossRef][PubMed] 29. Wang, Z.; Tan, B.; Hussain, I.; Schaeffer, N.; Wyatt, M.F.; Brust, M.; Cooper, A.I. Design of polymeric stabilizers for size-controlled synthesis of monodisperse gold nanoparticles in water. Langmuir 2007, 23, 885–895. [CrossRef][PubMed] 30. Huang, X.; Luo, Y.; Li, Z.; Li, B.; Zhang, H.; Li, L.; Majeed, I.; Zou, P.; Tan, B. Biolabeling hematopoietic system cells using near-infrared fluorescent gold nanoclusters. J. Phys. Chem. C 2011, 115, 16753–16763. [CrossRef] 31. Schaeffer, N.; Tan, B.; Dickinson, C.; Rosseinsky, M.J.; Laromaine, A.; McComb, D.W.; Stevens, M.M.; Wang, Y.Q.; Petit, L.; Barentin, C.; et al. Fluorescent or not? Size-dependent fluorescence switching for polymer-stabilized gold clusters in the 1.1–1.7 nm size range. Chem. Commun. 2008, 34, 3986–3988. [CrossRef] [PubMed] 32. Liu, F.; Zhu, J.H.; Hou, Y.L.; Gao, S. Chemical synthesis of magnetic nanocrystals: Recent progress. Chin. Phys. B 2013, 22, 107503. [CrossRef] 33. Ito, D.; Yokoyama, S.; Zaikova, T.; Masuko, K.; Hutchison, J.E. Synthesis of ligand-stabilized metal oxide nanocrystals and epitaxial core/shell nanocrystals via a lower-temperature esterification process. ACS Nano 2014, 8, 64–75. [CrossRef][PubMed] 34. Rui, Y.-P.; Liang, B.; Hu, F.; Xu, J.; Peng, Y.-F.; Yin, P.-H.; Duan, Y.; Zhang, C.; Gu, H. Ultra-large-scale production of ultrasmall superparamagnetic iron oxide nanoparticles for t1-weighted mri. RSC Adv. 2016, 6, 22575–22585. [CrossRef] 35. Wu, J.; Shen, Y.; Jiang, W.; Jiang, W.; Shen, Y. Magnetic targeted drug delivery carriers encapsulated with pH-sensitive polymer: Synthesis, characterization and in vitro doxorubicin release studies. J. Biomater. Sci. Polym. Ed. 2016, 27, 1303–1316. [CrossRef][PubMed] 36. Zhuang, L.; Zhao, Y.; Zhong, H.; Liang, J.; Zhou, J.; Shen, H. Hydrophilic magnetochromatic nanoparticles with controllable sizes and super-high magnetization for visualization of magnetic field intensity. Sci. Rep. 2015, 5, 17063. [CrossRef][PubMed] 37. Gupta, J.; Mohapatra, J.; Bhargava, P.; Bahadur, D. A pH-responsive folate conjugated magnetic nanoparticle for targeted chemo-thermal therapy and mri diagnosis. Dalton Trans. 2016, 45, 2454–2461. [CrossRef][PubMed] 38. Hirt, A.M.; Sotiriou, G.A.; Kidambi, P.R.; Teleki, A. Effect of size, composition, and morphology on magnetic performance: First-order reversal curves evaluation of iron oxide nanoparticles. J. Appl. Phys. 2014, 115, 044314. [CrossRef] 39. Liu, G.; Gao, J.; Ai, H.; Chen, X. Applications and potential toxicity of magnetic iron oxide nanoparticles. Small 2013, 9, 1533–1545. [CrossRef][PubMed]

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